Compound active peptide with skin anti-aging, anti-inflammatory and soothing functions and application of compound active peptide

Abstract

The invention relates to the technical field of cosmetics, in particular to a compound active peptide with skin anti-aging, anti-inflammatory and soothing functions and application of the compound active peptide. The compound active peptide is prepared from acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl ester, copper peptide and decarboxylated carnosine hydrochloride. The compound peptide acts on dermal fibroblasts, so that expression of p16 and p21 genes is reduced, and cell cycle arrest is improved; the compound acts on epidermal keratinocytes and dermal fibroblasts and inhibits COX-2 gene expression; the compound peptide acts on epidermal keratinocytes and dermal fibroblasts, and the ROS level is reduced; the compound peptide acts on PC12 cells, inhibits secretion of acetylcholine, blocks transmission of neural signals, and plays a role in immediate wrinkle removal.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to a compound active peptide with skin anti-aging, anti-inflammatory and soothing properties and its applications. Background Technology

[0002] As the largest organ in the human body, the skin is the primary barrier against external pathogens and ultraviolet radiation. It also functions in thermoregulation and water metabolism. The epidermis, dermis, and dermo-epidermal junction (DEJ) work together to maintain the normal physiological structure and function of the skin. Ultraviolet radiation is the core external factor causing photoaging of the skin. Among them, UVB (λ=280-320nm) is particularly damaging to the skin. Long-term exposure can lead to dry skin, sensitivity, and increased wrinkles. The mechanism is that UVB induces DNA damage in skin cells, activates the expression of cellular aging markers p16 and p21 genes, and causes cell cycle arrest. At the same time, it promotes senescent cells to secrete aging-associated secretory phenotype (SASP) containing pro-inflammatory factors such as IL-6 and IL-1β. It can also induce chronic inflammation through the activation of Toll-like receptors (TLRs) and upregulation of cyclooxygenase-2 (COX-2) expression, accelerating collagen degradation in the dermis and exacerbating skin aging.

[0003] While current skin anti-aging solutions include topical anti-aging medications, antioxidants, and bioactive peptides, the field of bioactive peptides has significant limitations: individual active peptides often act on only a single target, resulting in limited efficacy and high costs; some products blindly pile on active peptides, neglecting the synergistic effects between ingredients, which actually reduces overall efficacy. Therefore, there is an urgent need to screen high-quality active peptide raw materials and scientifically compound them to develop a compound active peptide that can synergistically act on the epidermis and dermis, soothing skin redness, reducing irritation, and delaying aging, in order to meet the cosmetic industry's demand for precise anti-aging and anti-inflammatory soothing.

[0004] Chinese invention patent application CN119345060A discloses a compound peptide, composition, and application with anti-inflammatory and soothing effects. Although it proposes a compound of tripeptide-1, acetyl dipeptide-1, palmitoyl tetrapeptide-7, and palmitoyl tripeptide-8 in a ratio of 0.5-1.5:0.6-1.5:1-2:0.5-1.5, which has anti-inflammatory, soothing, and anti-wrinkle effects, this scheme does not cover key components such as acetyl hexapeptide-1, copper peptide, and decarboxylated carnosine hydrochloride, nor does it design a multi-target regulatory mechanism for the synergistic effect of epidermal keratinocytes and dermal fibroblasts. Therefore, it cannot simultaneously achieve the comprehensive effects of reducing ROS levels, inhibiting acetylcholine secretion, and downregulating p16 / p21 gene expression. Summary of the Invention

[0005] The first aspect of the present invention provides a compound active peptide with anti-aging, anti-inflammatory and soothing properties for the skin, comprising acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl ester, copper peptide and decarboxylated carnosine hydrochloride.

[0006] The weight ratio of acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl ester, copper peptide and decarboxylated carnosine hydrochloride is (2-6):(2-6):(0.5-1.5):(0.5-1.5):(6-10).

[0007] This invention has found that the weight ratio of acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl ester, copper peptide, and decarboxylated carnosine hydrochloride (2-6):(2-6):(0.5-1.5):(0.5-1.5):(6-10) can achieve synergistic effects through multi-target, cross-skin layer functional complementarity. Decarboxylated carnosine hydrochloride, as the core antioxidant component, targets and eliminates ROS free radicals in skin cells, reduces oxidative stress damage caused by ultraviolet radiation, and simultaneously helps improve cell cycle arrest, laying the foundation for anti-aging and anti-inflammatory effects. Acetyl hexapeptide-1 and palmitoyl tripeptide-5 form an anti-aging synergy; the former blocks nerve signal transmission by inhibiting acetylcholine secretion in PC12 cells, rapidly improving dynamic wrinkles, while the latter delays cell aging and promotes collagen regeneration by downregulating the expression of p16 and p21 genes in dermal fibroblasts. These two ingredients target immediate wrinkle reduction and long-term anti-aging needs, respectively. Acetyl dipeptide-1 cetyl ester and copper peptide focus on anti-inflammatory, soothing, and anti-aging synergistic effects. The former precisely inhibits the expression of the COX-2 gene in epidermal keratinocytes and dermal fibroblasts, reducing UV-induced chronic inflammation and skin redness and sensitivity. The latter, in conjunction with palmitoyl tripeptide-5, further reduces the expression of aging marker genes and helps repair the skin barrier. The five ingredients work together on key cells in the epidermis and dermis, forming a closed loop of antioxidant damage reduction, inhibition of aging markers to delay cell aging, blocking of nerve signals to improve wrinkles, inhibition of inflammatory mediators to reduce sensitivity, and synergistic promotion of collagen to maintain skin elasticity. Inhibition of inflammation reduces collagen degradation, and antioxidants provide protection for cell anti-aging. The ingredients complement each other and have synergistic effects in specific proportions, ultimately achieving a comprehensive and highly effective synergistic anti-aging and anti-inflammatory soothing effect.

[0008] Optionally, the weight ratio of acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl ester, copper peptide and decarboxylated carnosine hydrochloride is (3-5):(3-5):(0.5-1.5):(0.5-1.5):(7-9).

[0009] Optionally, the content of acetyl hexapeptide-1 and palmitoyl tripeptide-5 in the compound active peptide is 15-30 wt%.

[0010] Optionally, the content of acetyl dipeptide-1 cetyl ester and copper peptide in the compound active peptide is 3-8 wt%.

[0011] Optionally, the content of acetyl dipeptide-1 cetyl ester and copper peptide in the compound active peptide is 6-8 wt%.

[0012] Optionally, the content of the decarboxylated carnosine hydrochloride in the compound active peptide is 35-60 wt%.

[0013] Optionally, the content of the decarboxylated carnosine hydrochloride in the compound active peptide is 35-40 wt%.

[0014] The second aspect of this invention provides an application of a compound active peptide with skin anti-aging, anti-inflammatory and soothing properties, which can be used in the preparation of medical aesthetic products or care products.

[0015] The concentration of the compound active peptide in medical aesthetic products or care products is 5-60 μM.

[0016] Optionally, the concentration of the compound active peptide in the medical aesthetic product or care product is 20-40 μM.

[0017] Optionally, the care products include at least one of toner, serum, lotion, cream, face cream, single-use ampoule, freeze-dried powder, facial mask liquid, facial mask powder, and frozen mask.

[0018] Beneficial effects 1. This invention, by limiting the weight ratio of acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl ester, copper peptide and decarboxylated carnosine hydrochloride to (2-6):(2-6):(0.5-1.5):(0.5-1.5):(6-10), can exert synergistic anti-aging, anti-inflammatory and soothing effects.

[0019] 2. The compound peptide of this invention acts on dermal fibroblasts, reduces the expression of p16 and p21 genes, and improves cell cycle arrest.

[0020] 3. The complex peptides of this invention act on epidermal keratinocytes and dermal fibroblasts to inhibit COX-2 gene expression.

[0021] 4. The compound peptides of this invention act on epidermal keratinocytes and dermal fibroblasts to reduce ROS levels.

[0022] 5. The compound peptide of this invention acts on PC12 cells, inhibits acetylcholine secretion, blocks nerve signal transmission, and exerts an immediate wrinkle-reducing effect. Attached Figure Description

[0023] Figure 1The examples and comparative examples illustrate the effect of peptides on p16 gene expression in photoaged HSF cells. From left to right, these represent the control group, model group, 5 μM peptide in comparative example 1, 5 μM peptide in comparative example 2, 20 μM peptide in comparative example 3, 40 μM peptide in comparative example 4, 5 μM complex peptide in example 1, 10 μM complex peptide in example 1, 20 μM complex peptide in example 1, 40 μM complex peptide in example 1, and retinoic acid.

[0024] Figure 2 The examples and comparative examples illustrate the effect of peptides on the expression level of p21 gene in photoaged HSF cells. From left to right, these represent the control group, model group, 5 μM peptide in comparative example 1, 5 μM peptide in comparative example 2, 20 μM peptide in comparative example 3, 40 μM peptide in comparative example 4, 5 μM complex peptide in example 1, 10 μM complex peptide in example 1, 20 μM complex peptide in example 1, 40 μM complex peptide in example 1, and retinoic acid.

[0025] Figure 3 To illustrate the effect of peptides in the examples and comparative examples on COX-2 gene expression in photoaged skin cells, the left graph corresponds to HaCaT cells, where the horizontal axis from left to right corresponds to the control group, model group, 5 μM peptide in comparative example 1, 5 μM complex peptide in example 1, 20 μM complex peptide in example 1, 40 μM complex peptide in example 1, and DXM (dexamethasone); the right graph corresponds to HSF cells, where the horizontal axis from left to right corresponds to the control group, model group, 5 μM peptide in comparative example 1, 5 μM complex peptide in example 1, 20 μM complex peptide in example 1, 40 μM complex peptide in example 1, and DXM.

[0026] Figure 4 To illustrate the effect of peptides in the examples and comparative examples on the level of reactive oxygen species (ROS) in HaCaT cells of aging skin, the left side is a flow cytometry histogram, from top to bottom corresponding to the control group, model group, 40 μM peptide in comparative example 4, 10 μM complex peptide in example 1, 20 μM complex peptide in example 1, 40 μM complex peptide in example 1, and vitamin C, respectively. The right side is a bar chart, from left to right corresponding to the control group, model group, 40 μM peptide in comparative example 4, 10 μM complex peptide in example 1, 20 μM complex peptide in example 1, 40 μM complex peptide in example 1, and vitamin C, respectively.

[0027] Figure 5To illustrate the effect of peptides in the examples and comparative examples on the level of reactive oxygen species (ROS) in the HSF of aging skin cells, the left side is a flow cytometry histogram, from top to bottom corresponding to the control group, model group, comparative example 4 peptide at a concentration of 40 μM, the complex peptide at a concentration of 10 μM in example 1, the complex peptide at a concentration of 20 μM in example 1, the complex peptide at a concentration of 40 μM in example 1, and vitamin C, respectively. The right side is a bar chart, from left to right corresponding to the control group, model group, comparative example 4 peptide at a concentration of 40 μM, the complex peptide at a concentration of 10 μM in example 1, the complex peptide at a concentration of 20 μM in example 1, the complex peptide at a concentration of 40 μM in example 1, and vitamin C, respectively.

[0028] Figure 6 To illustrate the inhibitory effect of the peptides in the examples and comparative examples on acetylcholine secretion in PC12 cells, the horizontal axis from left to right corresponds to the control group, the peptide at a concentration of 20 μM in comparative example 5, the complex peptide at a concentration of 10 μM in example 1, the complex peptide at a concentration of 20 μM in example 1, and the complex peptide at a concentration of 40 μM in example 1. Detailed Implementation

[0029] Example 1 A compound active peptide with anti-aging, anti-inflammatory and soothing properties for the skin is composed of the following peptides: acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl ester, copper peptide and decarboxylated carnosine hydrochloride; wherein the weight ratio of acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl ester, copper peptide and decarboxylated carnosine hydrochloride is 4:4:1:1:8.

[0030] Comparative Example 1 The peptide used is only acetyl dipeptide-1 cetyl ester.

[0031] Comparative Example 2 Only copper peptides are used.

[0032] Comparative Example 3 Only palmitoyl tripeptide-5 is used as the peptide.

[0033] Comparative Example 4 The peptides used are decarboxylated carnosine hydrochloride only.

[0034] Comparative Example 5 Only acetyl hexapeptide-1 is used as the peptide.

[0035] Performance testing methods and data The complex peptides of Example 1 were prepared into test samples at the following concentrations: 5 μM, 10 μM, 20 μM, and 40 μM. The peptide of Comparative Example 1 was prepared into a 5 μM test sample, the peptide of Comparative Example 2 was prepared into a 5 μM test sample, the peptide of Comparative Example 3 was prepared into a 20 μM test sample, the peptide of Comparative Example 4 was prepared into a 40 μM test sample, and the peptide of Comparative Example 5 was prepared into a 20 μM test sample. The above test samples were used for testing.

[0036] 1. The complex peptides act on photoaged skin cells, significantly reducing the expression levels of p16 and p21 genes. Cell cycle regulators p16 and p21 proteins regulate proliferation, differentiation, and senescence in skin cells. External stimuli such as ultraviolet radiation cause DNA damage in skin cells, activating p16 and p21 expression, inducing cell cycle arrest, and leading to senescence. This experiment examined the effects of complex peptides and single active peptides on the expression levels of p16 and p21 genes in photoaged HSF cells. The 5 μg / mL retinoic acid intervention group served as the control group (positive), while the model group received no peptides. Except for concentration, all other components were in DMEM complete culture medium.

[0037] Experimental methods: HSF cells were seeded into 12-well plates (2 × 10⁻⁶ cells per well). 5 (cells / well), after which the culture medium is discarded and the cells are irradiated with UVB lamps for 3 days (total irradiation dose 400 mJ / cm²). 2 Afterwards, different types and concentrations of active peptides and complex peptides were added and cultured for 48 hours. Cell RNA was extracted and the expression of p16 and p21 mRNA was detected.

[0038] Experimental results: like Figure 1 p16 gene expression results and Figure 2 The p21 gene expression results showed that, compared with the control group, the expression levels of p16 and p21 genes were significantly increased in the model group. 20-40 μM complex peptides, 5 μM copper peptides, and 20 μM palmitoyl tripeptide-5 significantly reduced p16 and p21 gene expression. The 40 μM complex peptides were more effective than decarboxylated carnosine hydrochloride, but the expression level in the 20 μM complex peptide intervention group was significantly higher than that in the 20 μM palmitoyl tripeptide-5 treatment group.

[0039] 2. The complex peptides act on photo-aged skin cells, inhibiting the expression of pro-inflammatory mediators. Chronic skin inflammation induced by ultraviolet (UV) radiation is one of the core mechanisms of photoaging. UV radiation stimulates skin cells to produce large amounts of SASPs, MMPs, and inflammatory cytokines, creating a chronic inflammatory environment that inhibits collagen synthesis and leads to its degradation. UV-induced DNA damage has long-term effects on inflammation-related genes such as COX-2. COX-2 synthesizes PGE2 and causes vasodilation, leading to symptoms such as burning and stinging, exacerbating the inflammatory response. PGE2 is closely related to neurogenic inflammation in sensitive skin. The complex peptide component contains anti-inflammatory and soothing peptides. This study examined the inhibitory effect of the complex peptides on pro-inflammatory mediators in photoaged skin cells and compared the efficacy with that of single anti-inflammatory and soothing peptides at the same concentration. Treatment with 10 μM dexamethasone served as a positive control.

[0040] Experimental methods: HaCaT cells or HSF cells were seeded into 12-well plates (2 × 10⁻⁶ cells / well). 5 (cells / well), after which the culture medium is discarded and the cells are irradiated with UVB lamps for 3 days (total irradiation dose 400 mJ / cm²). 2 Afterwards, different types and concentrations of active peptides and complex peptides were added and cultured for 48 hours. Cell RNA was extracted and mRNA expression was detected.

[0041] Experimental results: The results are as follows Figure 3 As shown, compared with the control group, the COX-2 gene expression level in the model group cells was significantly increased. 20-40 μM complex peptide and 5 μM acetyl dipeptide-1 cetyl ester significantly reduced COX-2 gene expression in photoaged HaCaT cells and HSF cells. The 5 μM complex peptide significantly inhibited COX-2 expression in HaCaT cells, but its inhibitory effect was less than that of acetyl dipeptide-1 cetyl ester.

[0042] 3. Complex peptides act on photo-aged skin cells, reducing ROS levels. Continuous exposure of the skin to endogenous stimuli (such as the mitochondrial respiratory chain) and exogenous stimuli (such as ultraviolet radiation) generates a large number of free radicals that attack intracellular biomolecules, leading to impaired cellular function and exacerbating oxidative stress and aging in skin cells. Antioxidants can neutralize free radicals, preventing oxidative damage to the skin and thus playing a certain anti-aging role. The complex peptide contains decarboxylated carnosine hydrochloride, which has the potential to target and scavenge free radicals. This study examined the effect of the complex peptide on ROS levels in photoaged HaCaT cells and HSF cells, and compared its antioxidant effect with that of 40 μM decarboxylated carnosine hydrochloride at the same concentration. 50 μM vitamin C intervention served as a positive control group.

[0043] Experimental methods: (1) HaCaT cells and HSF cells were seeded into 12-well plates, 2 × 10⁶ cells per well. 5Cells were cultured overnight to allow them to adhere. Except for the control group, cells in the other groups were irradiated with UVB to establish a photoaging model. After irradiation, cells were treated with different concentrations of compound peptides and 40 μM decarboxylated carnosine hydrochloride for 48 h, and treated with 50 μM vitamin C as a positive control group. The control and model groups were treated only with complete culture medium.

[0044] (2) After culture, discard the culture medium and wash with PBS. Refer to the instructions for reactive oxygen species detection, collect the cells, resuspend them in serum-free medium and wash once, then centrifuge again to collect the cell pellet. Prepare 10 μM MCFH-DA solution with fresh serum-free medium to resuspend the cells and incubate at 37°C in the dark for 30 min.

[0045] (3) After incubation, centrifuge to collect cell pellet, resuspend cells in PBS, and use flow cytometry to detect intracellular ROS content.

[0046] Experimental results: like Figure 4 Effects on reactive oxygen species (ROS) levels in HaCaT cells and Figure 5 The results regarding the effect on reactive oxygen species (ROS) levels in HSF cells showed that, compared with the control group, the ROS levels in the model group were significantly increased. Both 20-40 μM compound peptide and 40 μM decarboxylcarnosine hydrochloride significantly reduced ROS levels in HaCaT and HSF cells, and at the same concentration, there was no significant difference in ROS levels between the compound peptide treatment group and the decarboxylcarnosine hydrochloride treatment group. These results indicate that the antioxidant effect of the compound peptide is comparable to that of decarboxylcarnosine hydrochloride.

[0047] 4. Complex peptides inhibit acetylcholine secretion and block nerve signal transmission. Dynamic wrinkles form on the skin surface due to repeated folding caused by muscle contraction, leading to a decrease in epidermal smoothness. Therefore, reducing the intensity of facial muscle contraction and decreasing the depth and number of expression lines are important aspects of facial anti-aging. The complex peptide contains the neurotransmitter inhibitory peptide acetyl hexapeptide-1, which has immediate wrinkle-reducing potential. This experiment examined and compared the inhibitory effects of the complex peptide and acetyl hexapeptide-1 on acetylcholine secretion in PC12 cells.

[0048] Experimental methods: Logarithmic growth phase PC12 cells were seeded into 24-well plates at 5 × 10⁶ cells per well. 4 Cells were cultured overnight and then added to complete culture medium containing different concentrations of complex peptides and 20 μM acetyl hexapeptide-1. The control group was treated with only complete culture medium. After 48 h of culture, the culture medium was collected, the supernatant was collected by centrifugation, and the acetylcholine content was detected using an ELISA kit.

[0049] Experimental results: The results are as follows Figure 6As shown, compared with the control group, the acetylcholine content in the 20 μM acetyl hexapeptide-1 intervention group was significantly reduced. The acetylcholine inhibition rate of 40 μM complex peptide III was 18%, which was significant compared with the control group.

[0050] In summary, the compound peptides provided by this invention act on keratinocytes in the epidermis and fibroblasts in the dermis, and achieve multiple anti-aging, anti-inflammatory and soothing effects on the epidermis and dermis by influencing the expression levels of cellular aging marker genes, pro-inflammatory mediator genes, ROS levels, and cellular acetylcholine secretion.

Claims

1. A compound active peptide with skin anti-aging, anti-inflammatory and soothing properties, characterized in that, The acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl esters, copper peptide and decarboxy carnosine hydrochloride.

2. The complex active peptide with skin anti-aging, anti-inflammatory and soothing properties according to claim 1, characterized in that, The weight ratio of the acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl esters, copper peptide and decarboxy carnosine hydrochloride is (2-6):(2-6):(0.5-1.5):(0.5-1.5):(6-10).

3. The complex active peptide with skin anti-aging, anti-inflammatory and soothing properties according to claim 2, characterized in that, The weight ratio of the acetyl hexapeptide-1, palmitoyl tripeptide-5, acetyl dipeptide-1 cetyl esters, copper peptide and decarboxy carnosine hydrochloride is (3-5):(3-5):(0.5-1.5):(0.5-1.5):(7-9).

4. The complex active peptide with skin anti-aging, anti-inflammatory and soothing properties according to claim 3, characterized in that, The content of the acetyl hexapeptide-1 and palmitoyl tripeptide-5 in the complex active peptide is 15-30wt%.

5. The complex active peptide with skin anti-aging, anti-inflammatory and soothing properties according to claim 3, characterized in that, The content of the acetyl dipeptide-1 cetyl esters and copper peptide in the complex active peptide is 3-8wt%.

6. The complex active peptide with skin anti-aging, anti-inflammatory and soothing properties as claimed in claim 3, wherein, The content of the decarboxy carnosine hydrochloride in the complex active peptide is 35-60wt%.

7. Use of the complex active peptide having skin anti-aging, anti-inflammatory and soothing properties according to any one of claims 1 to 6, characterized in that, The complex active peptide is applied to the preparation of medical and beauty products or care products.

8. Use of the complex active peptide having skin anti-aging, anti-inflammatory and soothing properties according to claim 7, characterized in that, The concentration of the complex active peptide in the medical and beauty products or care products is 5-60μM.

9. Use of the complex active peptide having skin anti-aging, anti-inflammatory and soothing properties according to claim 8, characterized in that, The concentration of the complex active peptide in the medical and beauty products or care products is 20-40μM.

10. Use of the complex active peptide having skin anti-aging, anti-inflammatory and soothing properties according to claim 7, characterized in that, The care products include at least one of toner, essence, emulsion, cream, face cream, secondary polishing stock solution, freeze-dried powder, mask liquid, mask powder and frozen mask.

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